Prothrombin is a vitamin K-dependent plasma protein involved in blood coagulation. Prothrombin has a molecular weight of about 72,000 and is a calcium-binding protein that undergoes a conformational transition in the presence of calcium. When activated, prothrombin undergoes proteolytic cleavage at two sites to yield a two-chain molecule linked by a disulfide bond. This new product is thrombin. Thrombin converts fibrinogen to fibrin to produce the initial visible manifestation of coagulation, the soluble fibrin clot.
The proteolytic activation of prothrombin to form thrombin is a critical step in normal hemostasis. Prothrombin is synthesized in the liver where a prothrombin precursor undergoes posttranslational modification to yield the normal, functional form of prothrombin, which is known as "native prothrombin" and contains .gamma.-carboxyglutamic acid. A nonfunctional, deficient form of prothrombin is known as descarboxyprothrombin, and is not activated to form thrombin.
In the presence of vitamin K antagonists, such as sodium warfarin (also known as COUMADIN.RTM.), or in the absence of vitamin K, prothrombin activity in the blood can be significantly diminished. Severe liver disease also can be associated with low plasma prothrombin activity. Thus, impaired synthesis of proteins (liver disease), inadequate supplies of vitamin K (vitamin K deficiency), or drugs that inhibit the action of vitamin K (sodium warfarin) lead to diminished plasma prothrombin activity in humans and other mammals.
COUMADIN.RTM. is used as an oral anticoagulant in the therapy or prevention of thrombotic disease. COUMADIN.RTM. lowers the activity of vitamin K-dependent blood coagulation proteins such as prothrombin. The appropriate COUMADIN.RTM. dose is established by monitoring the prothrombin time, or a one-stage coagulation assay. The appropriate dose also can be monitored by direct measurement of prothrombin coagulant activity using prothrombin deficient substrate plasma, but this is not a common procedure.
The prothrombin time, however, is a test influenced by blood level concentrations of two additional proteins, specifically, Factor X and Factor VII. An alternate method that is not in widespread use is to measure the level of prothrombin in the blood.
Direct assays for prothrombin coagulant activity are carried out by mixing test plasma with varying amounts of prothrombin-deficient plasma, followed by clotting and comparison with a standard curve. Echis carinatus assays using snake venom are used to measure total prothrombin, i.e., the sum of native prothrombin and descarboxyprothrombin. For individuals undergoing therapy using sodium warfarin, descarboxyprothrombin is present in the plasma, together with native prothrombin. The above-described assay for total prothrombin cannot differentiate between native prothrombin and descarboxyprothrombin, and, therefore, cannot be used to measure native prothrombin concentration in individuals undergoing sodium warfarin therapy.
Yamada et al., J. Biol. Chem., 271(9), pp. 5200-5207 (1996), discloses the characterization of carinactivase-1 (CA-1), a prothrombin activator present in the venom of certain subspecies of Echis carinatus. This publication disclosed that CA-1 can be used to assay the plasma of individuals using a vitamin K antagonist, e.g., an oral anticoagulant. The disclosed assay requires mixing the test plasma with varying amounts of prothrombin-deficient plasma.
The current method of measuring plasma prothrombin levels requires making several dilutions of the plasma of interest, or a reference plasma, then mixing these diluted plasmas with a plasma deficient in prothrombin. Next, the prothrombin time of the mixtures is determined, and the prothrombin concentration of the plasma of interest is calculated from a standard curve established by assaying the reference plasma. Such assays typically are performed by specially trained laboratory technicians using coagulation machines. The current assay method for prothrombin, therefore, is labor intensive, expensive, and susceptible to inaccurate results due to technician error resulting from numerous manipulative steps.
It would be advantageous, therefore, to provide a method of determining plasma prothrombin concentration within minutes after adding a reagent to undiluted plasma or whole blood, without dilution or other special preparation of the sample, and without the use of prothrombin-deficient plasma. Such a method would be useful in monitoring prothrombin levels in individuals taking oral anticoagulants, suffering from liver disease, or whose prothrombin levels are measured for other clinical purposes.
Therefore, for an individual to monitor anticoagulant therapy, or to monitor a treatment to overcome a specific blood factor deficiency, an accurate and sensitive assay of undiluted whole blood and blood plasma for prothrombin is needed. The assay should permit the rapid detection and measurement of prothrombin in the test sample such that a correct medical treatment is implemented, monitored, and maintained. In addition, it would be advantageous for the assay method to utilize a dry phase test strip for the easy and economical determination of prothrombin in undiluted whole blood or plasma.
Furthermore, any method of assaying for prothrombin in whole blood or plasma should yield accurate, trustworthy, and reproducible results by utilizing a composition that undergoes a detectable transition as a result of an interaction with normal prothrombin, and not as a result of a competing interaction with a deficient form of prothrombin, i.e., descarboxyprothrombin. Moreover, it would be advantageous if the assay method for prothrombin is suitable for use in dry phase reagent test strips for the rapid, economical, and accurate determination of prothrombin in whole blood or plasma.
Therefore, in order to monitor the course of medical treatment for anticoagulants to determine the effectiveness of the treatment, or to overcome a specific blood factor deficiency, simple, accurate, and inexpensive detection assays for prothrombin are needed. Furthermore, the assay method should obviate dilution of the test sample and the use of prothrombin-deficient plasma.
Present-day assays for prothrombin do not assay undiluted blood or plasma, and have the disadvantages of requiring the use of prothrombin-deficient plasma. Surprisingly and unexpectedly, the composition and method of the present invention provide a composition that can be used in the assay of undiluted whole blood or plasma for prothrombin, without the use of a prothrombin-deficient plasma. By providing an accurate method of determining the concentration of prothrombin in a test sample, in an easy to use format, such as a dip-and-read test strip, the assay can be performed by laboratory personnel, or at home, to afford immediate and trustworthy test results. In addition, a test strip method can be performed to more precisely monitor the level of prothrombin in undiluted whole blood or plasma and/or the success of the medical treatment the individual is undergoing.